Today, antibodies are widely used agents in the field of medicine and research. In medicine, they find application in many different fields. For example, antibodies are used as labeling agents for detecting certain markers which allow the diagnosis and/or prognosis of diseases or the determination of specific body parameters such as, for example, the presence or concentration of certain hormones.
Furthermore, antibodies are also used as therapeutic agents in the treatment and prophylaxis of a variety of diseases such as cancer, cardiovascular diseases, inflammatory diseases, macular degeneration, transplant rejection, multiple sclerosis, and viral infections. In these therapies, the antibody may possess therapeutic activity on it own, for example by blocking receptors or messenger molecules, thereby inhibiting their disease-relevant functions, or by recruiting and activating components of the patient's immune system. Alternatively, the antibody may be coupled to another agent having therapeutic activity. In particular in the treatment of cancer and infections, said further agent has cell-killing activity and may be, for example a radioisotope or a cytotoxin. In another application, antibodies may be used to passively immunize a patient by transferring suitable antibodies into the patient's circulation.
Specific antibodies are produced by injecting an antigen into a mammal, such as a mouse, rat, rabbit, goat, sheep, or horse. Blood isolated from these animals contains polyclonal antibodies directed against said antigen in the serum. To obtain an antibody that is specific for a single epitope of an antigen, antibody-secreting lymphocytes are isolated from the animal and immortalized by fusing them with a cancer cell line, resulting in hybridoma cells. Single hybridoma cells are then isolated by dilution cloning to generate cell clones that all produce the same monoclonal antibody.
However, in therapeutic applications these monoclonal antibodies have the problem that they are derived from animal organisms and differ in their amino acid sequence from human antibodies. The human immune system hence recognizes these animal antibodies as foreign and rapidly removes them from circulation. Furthermore, systemic inflammatory effects may be caused. A solution to this problem is the replacement of certain constant parts of the monoclonal antibody with corresponding parts of a human antibody. If only the heavy and light chain constant regions are replaced, a chimeric antibody is obtained, while the additional replacement of the framework regions of the heavy and light chain variable regions results in so called humanized antibodies.
In research, purified antibodies are used in many applications. They are most commonly used to identify and locate biological molecules such as in particular proteins. The biological molecules may either be detected after they have been isolated, for example to determine their presence, concentration, integrity or size. On the other hand, they may be detected in cellular or tissue samples, for example to determine their presence or location. Furthermore, antibodies are used in isolation procedures of specific biological substances, in particular proteins, wherein the antibody specifically separates the biological substance of interest from the sample containing it.
In all these applications, a tight binding and specific recognition of the antigen is of vital importance for the antibody used. Thereby, higher activity and less cross-reactivity, in particular less adverse side effects in therapeutic applications, are obtained. However, during humanization of monoclonal antibodies, often the affinity and specificity of the engineered antibody is decreased.
An interesting and important group of antibodies are those directed against epidermal growth factor receptors (EGFR). The EGF receptor is a receptor tyrosine kinase which is anchored in the plasma membrane. The extracellular domain binds to epidermal growth factor which results in dimerization of the receptor and stimulation of its intracellular protein-tyrosine kinase activity. The signal transduction cascades initiated by the active receptor dimer control cell migration, adhesion, and proliferation.
Overexpression of EGFR or overactivity has been found in a number of cancers, including lung cancer, anal cancers, and glioblastomas. Mutations, amplifications or misregulations of EGFR or family members are implicated in about 30% of all epithelial cancers and are associated with a poor prognosis.
Antibodies, especially monoclonal antibodies, raised against EGFR have been established as anti-tumor agents. Such antibodies may compete with the EGFR ligands such as EGF and TGFα in binding to the receptors, thereby inhibiting the growth of tumors that express the receptor. Furthermore, the antibodies may inhibit the growth of tumors immunologically through antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In another approach, toxins are conjugated to the antibodies. The antibody portion directs the conjugate to the tumor, which is then killed by the toxin portion.
Several antibodies directed against EGFR are known in the art. Some of them are already approved for medical applications. However, a disadvantage of using murine monoclonal antibodies in human therapy is the possibility of a human anti-mouse antibody (HAMA) response due to the presence of mouse Ig sequences. This disadvantage can be minimized by replacing the entire constant region of a murine (or other non-human mammalian) antibody with that of a human constant region (chimerization). The chimerization process can be made more effective by also replacing the framework regions of the variable regions of a murine antibody with the corresponding human sequences (humanization). The humanized antibody is less immunogenic (i.e. elicits less of a HAMA response) as more murine sequences are replaced by human sequences.
Unfortunately, a humanized antibody often has a lower affinity and specificity for its target antigen than the corresponding non-human or chimeric antibody. This, as the overall three-dimensional structure of the variable regions and in particular the conformation and orientation of the complementarity determining regions (CDRs) may be altered by the replacement of the framework regions.
Therefore, there is a need in the art to provide humanized antibodies, in particular humanized anti-EGFR antibodies, in particular humanized versions of Cetuximab, which have an antigen binding affinity and antigen specificity similar to that of the corresponding murine or chimeric antibody.